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M O D E S O F T R A N S C R I P T I O N A L R E G U L AT I O N

RNA-mediated epigenetic regulation

of gene expression
Daniel Holoch and Danesh Moazed
Abstract | Diverse classes of RNA, ranging from small to long non-coding RNAs, have
emerged as key regulators of gene expression, genome stability and defence against
foreign genetic elements. Small RNAs modify chromatin structure and silence
transcription by guiding Argonaute-containing complexes to complementary
nascent RNA scaffolds and then mediating the recruitment of histone and DNA
methyltransferases. In addition, recent advances suggest that chromatin-associated long
non-coding RNA scaffolds also recruit chromatin-modifying complexes independently
of small RNAs. These co‑transcriptional silencing mechanisms form powerful RNA
surveillance systems that detect and silence inappropriate transcription events, and
provide a memory of these events via self-reinforcing epigenetic loops.

RNA interference
In many organisms, intergenic or antisense transcrip-
(RNAi). Broadly refers to RNA tion gives rise to different classes of small RNAs and
silencing pathways that use long non-coding RNAs (lncRNAs) that have emerged
Argonaute and PIWI proteins as key regulators of chromatin structure in eukaryotic
and small RNAs to silence gene
cells1,2. In addition to their roles in RNA degradation
expression.
and translational repression, small RNAs modify chro-
Small interfering RNAs matin and target gene expression via RNA interference
(siRNAs). 22–24‑nucleotide (RNAi) pathways3–11 (BOX 1). In many instances, nuclear
small RNAs that are generated RNAi pathways mediate histone or DNA methylation
from longer double-stranded
RNA precursors by the
events that repress transcription. Studies of the mustard
ribonuclease Dicer. plant Arabidopsis thaliana6–9,12 first demonstrated that
post-transcriptional gene silencing, and the accompanying
DNA methylation of target loci, correlated with the pro-
duction of small interfering RNAs (siRNAs) and thus linked
RNA-directed DNA methylation to the RNAi pathway,
which had previously been described in Caenorhabditis
elegans13. Studies in the fission yeast Schizosaccharomyces
pombe, the ciliate protozoa Tetrahymena thermophila,
as well as in animal germline and somatic cells, then
revealed a general role for RNAi and related mechanisms
Howard Hughes Medical in heterochromatin formation or DNA methylation10,14–17
Institute, Department of Cell (see Supplementary information S1 (figure)).
Biology, Harvard Medical
RNA also regulates chromatin modifications and
School, 240 Longwood
Avenue, Boston, structure through pathways that do not involve RNAi;
Massachusetts 02115, USA. some lncRNAs, and even some mRNAs, seem to contain
Correspondence to D.M.  signals that recruit chromatin-modifying complexes
e-mail: danesh@hms.harvard. independently of small RNAs18. Early examples include
edu
doi:10.1038/nrg3863
X inactive specific transcript (XIST), which coats the
Published online entire inactive X chromosome in female mammals,
2 January 2015 and RNA on the X 1 (roX1) and roX2, which coat the
X chromosome in male flies, ultimately leading to
increased transcription 19–21. More recently, a large
number of other lncRNAs have been shown to act at
a gene-specific level, rather than at a chromosomal
level, to either activate or silence transcription18,22 (see
Supplementary information S1 (figure)).
A unifying mechanism by which small RNAs and
lncRNAs modify chromatin structure and silence tran-
scription is the formation of RNA scaffolds. Although
the machineries that use RNA scaffolds have greatly
diverged throughout evolution, a number of key simi-
larities enable us to define the common themes and
principles that are conserved in eukaryotes from fission
yeast to mammals.
In this Review, we discuss recent progress in our
understanding of the role of RNA in genome regulation,
focusing on the roles of different classes of chromatin-
bound RNAs as scaffolds for chromatin-modifying
complexes. Moreover, we review recent mechanistic
insights into how small-RNA amplification loops are
coupled to histone or DNA methylation to form self-
reinforcing positive feedback systems that maintain
epigenetic states. First, we discuss how small RNAs and
Argonaute (AGO) complexes are assembled and how they
target specific chromatin regions for silencing, focusing
on the better understood S. pombe and A. thaliana sys-
tems where distinct mechanisms have been elucidated
by which siRNAs and histone or DNA methylation
events form self-reinforcing epigenetic loops. Second,
we review nuclear small-RNA silencing pathways in

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Heterochromatin other model systems, including C. elegans, Drosophila  results, it was suggested that RNAi has an important
Regions of chromatin that melanogaster and mammals, highlighting conservation
retain the condensed and divergence in the roles of these pathways in gene and
appearance of mitotic genome regulation. Finally, we discuss the mechanisms
chromosomes throughout the
cell cycle. Heterochromatic
by which lncRNAs and mRNAs interact with RNA
regions are associated processing and chromatin-modifying machineries
with repressive histone independently of RNAi pathways.
modifications and
structural proteins, and are
RNAi-mediated heterochromatin assembly
transcriptionally silent.
RNAi-mediated transcriptional gene silencing is best
Argonaute understood in S. pombe, in which many basic principles
(AGO). A family of proteins of the pathway were first deciphered. S. pombe contains
that bind to small RNAs a single gene each for Ago (ago1+), Dicer (dcr1+) and
and that are conserved in all
domains of life. They mediate
RNA-dependent RNA polymerase (RdRP; rdp1+) (BOX 2).
target recognition via Deletion of any of these genes was shown to result in loss
base-pairing interactions of heterochromatic gene silencing at pericentromeric DNA
between their bound small repeat regions and a reduction in the levels of histone
RNA and complementary
H3 lysine 9 (H3K9) methylation, a conserved marker
coding or non-coding RNAs.
of heterochromatin in organisms ranging from yeast to
plants and mammals10. Additionally, early sequencing
experiments detected small RNAs that mapped to the
pericentromeric repeat regions, and Rdp1 was found
to associate with centromeric DNA10,11. Based on these
Box 1 | Small-RNA silencing pathways
RNA interference (RNAi) is used broadly to refer to various RNA silencing pathways
that use small RNAs, together with a member of the conserved Argonaute (AGO) and
PIWI family of proteins, to target genes for inactivation at the post-transcriptional or
transcriptional levels1,140. Classical RNAi is triggered by long double-stranded RNA
(dsRNA) precursors, which are processed to 22–23‑nucleotide duplex small interfering
RNAs (siRNAs) with 2‑nucleotide 3ʹ overhangs by the RNase III ribonuclease
Dicer141–145. siRNA duplexes contain 5ʹ monophosphate and 3ʹ OH groups, and are
loaded onto AGO family proteins, which associate with the siRNA 5ʹ monophosphate-
containing nucleotide via their middle (MID) domain, and with the siRNA 3ʹ nucleotide
via their PIWI–AGO–ZWILLE (PAZ) domain146–148. The arrangement and specificity of
the MID and PAZ domains in AGO proteins allow them to associate with small RNAs
of specific sizes with distinct termini. siRNAs guide AGO proteins and their associated
complexes to complementary RNAs, which are then targeted for degradation,
translational repression or transcriptional silencing3–5. In addition to the MID and PAZ
domains, AGO proteins contain an RNase H‑like domain that can cleave or slice target
RNAs, promoting their degradation149. In some organisms, this slicer activity is also
critical for the release of one of the two AGO-bound siRNA strands and the conversion
of duplex siRNA to mature single-stranded siRNA150–153. In Schizosaccharomyces pombe,
plants, Tetrahymena thermophila and Caenorhabditis elegans, siRNA-programmed
AGO recruits an RNA-dependent RNA polymerase (RdRP), which uses the targeted
RNA as a template to synthesize a dsRNA substrate for Dicer, thereby amplifying
siRNAs and the RNAi response27,154–156.
The genomes of Drosophila melanogaster and mammals do not seem to encode
RdRPs. However, these organisms harbour another class of small RNAs,
PIWI-interacting RNAs (piRNAs), which mediate RNA degradation in the cytoplasm and
DNA or histone methylation in the nucleus157–160. Metazoan piRNAs originate from
single-stranded RNA precursors, and their amplification in the D. melanogaster and
mammalian germ lines involves the ‘ping-pong’ cycle whereby piRNA-guided cleavage
of complementary RNAs by one PIWI paralogue generates the 5ʹ ends of new piRNAs
that are loaded onto another paralogue, and vice versa, in a process that degrades
transposon mRNAs84,161,162. It is proposed that piRNA pools are initially derived from
random sampling of the transcriptome and then selectively enriched by ping-pong
amplification for sequences corresponding to transposons that are actively transcribed
and that are able to contribute substrates to the cycle85. This idea is reminiscent of
primal RNAs (priRNAs) in S. pombe. priRNAs also result from cellular RNA sampling by
Ago1, but they only direct small-RNA amplification at loci where antisense RNA targets
are available163. This leads to repeat-specific RNAi-dependent siRNA accumulation163.
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© 2015 Macmillan Publishers Limited. All rights reserved
role in the initiation of heterochromatin formation
and its subsequent maintenance at pericentromeric
DNA repeats via the Rdp1‑mediated recruitment of
H3K9 methylation. However, later studies indicated
that a small-RNA-programmed Ago1 complex medi-
ates the targeting of specific chromosome regions and
recruitment of H3K9 methylation23 (see below).
The RITS complex and the emergence of the ‘nascent
transcript’ model. A physical connection between the
RNAi pathway and heterochromatin was established by
the purification of Chp1, a chromodomain protein that is
required for silencing the same heterochromatic regions
targeted by RNAi23,24. Chp1 was found to be a component
of an RNA-induced transcriptional silencing (RITS) complex
that also contains Ago1 and heterochromatic small RNAs
(BOX 2). The third RITS complex subunit, Tas3, is also
required for silencing and contains a conserved Ago-
binding GW domain23,25,26. The RITS complex associates
with the RNA-dependent RNA polymerase complex
(RDRC), which includes Rdp1, the helicase Hrr1 and
the non-canonical poly(A) polymerase Cid12 (REF. 27).
Both complexes associate not only with heterochromatin
and one another, but also with non-coding pericentro-
meric RNAs27. The functional importance of this RNA
association is made clear by the observation that ectopic
tethering of the RITS complex to a euchromatic mRNA
triggers H3K9 methylation at its site of transcription28.
Together, these results led to the development of the
‘nascent transcript’ model for RNAi-dependent hetero-
chromatin assembly 27,28 (FIG. 1). Chromatin-associated
RNAs are thought to act as scaffolds for the cooperative
assembly of complex machineries that couple small-RNA-
mediated recognition to chromatin modifications. This
co-transcriptional gene silencing mechanism contains
two primary components28–31. First, the nascent tran-
script is degraded by both RNAi-dependent and RNAi-
independent mechanisms, with RNAi-independent
mechanisms involving TRAMP and exosome complexes29.
Second, RNAi-dependent H3K9 methylation leads to
hetero­chromatin formation and transcriptional gene
silencing30,31. This small-RNA-based targeting strategy for
heterochromatin assembly is in contrast with that used
by the distant fungal relative Saccharomyces cerevisiae,
in which specificity is instead determined by site-specific
DNA-binding proteins (reviewed in REF. 32).
In addition to the RDRC and the RITS complex, the
nascent transcript heterochromatin assembly platform
also includes the Clr4–Rik1–Cul4 (CLRC) complex, the
Clr4 subunit of which is the sole H3K9 methyltrans-
ferase in S. pombe33–37. The CLRC complex subunit Rik1
associates with both the RDRC and the RITS complex,
and the peripheral CLRC member Stc1 also provides
a link to the RNAi machinery via its interaction with
Ago1, although it seems to be dispensable for the inter-
action of the CLRC complex with the RITS complex
subunit Tas3 (REFS 38−40). These physical connections
suggest the existence of a feedback loop in which the
activities of the RITS complex, the RDRC and the CLRC
complex reinforce each other (FIG. 1) (see below).
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Box 2 | Heterochromatic small RNAs

Heterochromatic gene
silencing
Silencing of gene expression
within heterochromatin. It was
originally thought to exclusively
involve transcriptional gene
silencing mechanisms, but
recent findings indicate that
co‑transcriptional degradation
of nascent RNA, or
co‑transcriptional gene
silencing, also play important
parts in silencing.
Pericentromeric DNA repeat
A repeated DNA sequence that
surrounds the centromeres of
most eukaryotic chromosomes.
These repeats are assembled
into heterochromatin, which
has been demonstrated
to have roles in cohesin
recruitment in fission yeast
and mammals, and de novo
centromere assembly in fission
yeast.
The discovery of small RNAs derived from the
pericentromeric repeats of the fission yeast
Schizosaccharomyces pombe marked the first example of Pol II
heterochromatic small RNAs in any organism11, and cen repeat
studies of S. pombe have remained useful for uncovering the Pol II
modes of biogenesis of this small-RNA class. Non-coding
transcripts generated from pericentromeric DNA repeats
(cen) act as the precursors for heterochromatic small
interfering RNAs (siRNAs) in S. pombe. Initially, it was
hypothesized that heterochromatic small RNAs arise from
Dicer 1 (Dcr1) cleavage of double-stranded RNA (dsRNA) dsRNA priRNA
formed by the base-pairing of complementary
Dcr1
pericentromeric transcripts. According to this hypothesis,
the RNA-dependent RNA polymerase Rdp1 amplifies the
siRNA pool by generating additional Dcr1 substrates10. Primary siRNA
In this scenario, heterochromatic small RNA levels are
expected to be higher in cells lacking Rdp1, in which
pericentromeric transcripts from opposite strands can still
hybridize and undergo Dcr1 processing, than in cells lacking
Dcr1. Initially, no differences in small RNA levels in rdp1− Ago1 Ago1
and dcr1− cells were detected on northern blots or in early
small-RNA sequencing experiments27,163. However, recent
advances in increasing the depth of sequencing have enabled Triman
the detection of a small population of Dcr1‑dependent,
Rdp1‑independent heterochromatic small RNAs known as Rdp1
primary siRNAs, which are thought to arise from the Ago1
base-pairing and Dcr1 processing of bidirectionally
synthesized transcripts164. Dcr1 cleavage
Dcr1 can limit the biogenesis of primary siRNAs, as and siRNA amplification
overexpression of Dcr1 not only increases primary siRNA
levels but also induces siRNA production from convergently H3K9 methylation
transcribed loci genome-wide164. Thus, limited Dcr1
availability is likely to represent an adaptation that restricts cen repeat
heterochromatic small-RNA biogenesis to a subset of loci
with high levels of bidirectional transcription. The low
abundance of Dcr1 and primary siRNAs is compensated for by the Rdp1‑mediated generation of secondary
heterochromatic siRNAs and also by the cooperation of the two enzymes — Dcr1 associates with Rdp1 Nature
andReviews | Genetics
stimulates its
dsRNA synthesis activity50. Consistent with an adaptive relationship between low Dcr1 abundance and Rdp1 activity,
overexpression of Dcr1 suppresses, to a large extent, the requirement for Rdp1 and for Dsh1, a factor required for Dcr1–
Rdp1 association, in pericentromeric silencing and histone H3 lysine 9 (H3K9) methylation164,165. These observations also
rule out a critical role for Rdp1 in the direct recruitment of H3K9 methylation activity to chromatin as previously proposed10.
Finally, a class of Dcr1‑independent heterochromatic small RNAs known as primal RNAs (priRNAs), which are capable of
directing H3K9 methylation in an Ago1‑dependent manner, has been discovered (see the figure)163. This finding, together
with the detection of primary siRNAs164, suggests that self-reinforcing feedback loops involving small RNAs and chromatin
modification (BOX 3) may be nucleated by their small RNA components. In support of this idea, priRNAs and siRNAs, which
are both trimmed to their mature length by the 3ʹ exonuclease Triman, are required for the maintenance of
RNAi-dependent facultative heterochromatin islands and de novo establishment of constitutive pericentromeric
heterochromatin (see the figure)166.
Pol II, RNA polymerase II.

RNA-induced transcriptional
silencing
(RITS). A protein complex
first identified in
Schizosaccharomyces pombe.
In addition to Argonaute 1, the
RITS complex contains a GW
domain protein, Tas3, and a
chromodomain protein, Chp1,
which tether the complex
to the chromosome via
interactions with nascent
long non-coding RNAs and
nucleosomes with methylated
histone H3 lysine 9.
The central role of nascent RNAs in the recruit-
ment of RNAi and histone-modifying activities has
prompted investigations of the interactions between
RNA-mediated silencing and other nuclear processes in
S. pombe. The CLRC complex components Raf2 (also
known as Cmc2) and Rik1 were recently found to asso-
ciate with Cdc20 (the catalytic subunit of the leading-
strand DNA polymerase‑ε) and Mms19 (a conserved
regulator of the general transcription factor TFIIH41),
suggesting the coordination of DNA replication with
RNAi-dependent release of RNA polymerase II (Pol II)
in the inheritance of heterochromatin42. However, this
Raf2–Rik1 complex seems to be distinct from the CLRC
complex, and direct evidence of cooperation between
the RNAi and DNA replication machineries is lacking.
It has also been proposed that the splicing machinery
contributes to heterochromatic silencing by directly
interacting with the RDRC and the nascent transcript
platform to promote siRNA biogenesis43,44. However,
earlier biochemical purifications demonstrated that
the RDRC subunit Cid12 forms RDRC-independent
complexes with splicing factors27. Moreover, the RNAi
defect found in several splicing mutants can be par-
tially rescued by the introduction of cDNAs that encode

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siRNA ARC
Tas3 Ago1
RITS
Chp1
Tas3 Ago1
Chp1
Dcr1
RDRC TRAMP and
exosome
Ers1 Tas3 Ago1
Swi6 Chp1
H3K9me2 or
H3K9me3
Centromeric
repeats Pol II
Pol II
Chp1 Chp2 Swi6
Ago1 Tas3 Stc1
RITS Raf1 Rik1 Clr4 SHREC
Raf2 Cul4 CLRC
lncRNA
Figure 1 |  The ‘nascent transcript’ model and a self-reinforcing
Natureepigenetic loop in
Reviews | Genetics
S. pombe. In Schizosaccharomyces pombe, the RNA-induced transcriptional silencing
(RITS) complex establishes a physical connection between small interfering RNAs
(siRNAs) and heterochromatin by targeting a nascent transcript, and forms the basis of
a self-sustaining feedback mechanism that couples siRNA production to chromatin
modification. A siRNA-targeted centromeric long non-coding (lncRNA) bound to the
RITS complex becomes a template for double-stranded RNA (dsRNA) synthesis by
the RNA-dependent RNA polymerase complex (RDRC, which is composed of Rdp1,
Hrr1 and Cid12) and generation by Dicer 1 (Dcr1) of new siRNAs, leading to further
targeting of the RITS complex after passage of Argonaute (Ago) through the ARC (Ago
siRNA chaperone) complex. The Chp1 subunit of the RITS complex anchors the
complex onto nucleosomes with histone H3 lysine 9 (H3K9) methylation, and
the RITS complex recruits the Clr4–Rik1–Cul4 (CLRC, of which Clr4 is the
methyltransferase) complex via Rik1 and Stc1 to promote the further spread of
H3K9 methylation. The heterochromatin protein 1 (HP1) homologue Swi6 binds to
methylated H3K9 and promotes RDRC recruitment and siRNA biogenesis via the
silencing factor Ers1. Swi6, and particularly the other HP1 protein Chp2, help to
restrict RNA polymerase II (Pol II) access by recruiting the Snf2–histone deacetylase
repressor complex (SHREC ). The TRAMP non-canonical poly(A) polymerase and the
exosome also contribute to silencing. Together, the RITS complex and the nascent
lncRNA transcript provide a hub for the assembly of machineries that make siRNAs,
modify histones and silence gene expression. me2, dimethylation.
RNAi components45, suggesting that the effects of splic-
ing mutations on heterochromatic silencing are at least
partially due to improper splicing of pre-mRNAs that
encode RNAi proteins.
siRNAs form self-reinforcing epigenetic loops with DNA
and histone methylation. The observation that silent
states of gene expression and their associated chromatin
modifications can spread along DNA in cis and persist
throughout cell division32 led to the idea that positive
feedback loops might form the molecular basis for epige-
netic memory. Linking the recognition of signals — such
as covalent histone modifications, DNA methylation or
small RNAs — to the generation of new signals would
ensure the stable propagation of silent states46–48 (BOX 3).
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© 2015 Macmillan Publishers Limited. All rights reserved
Indeed, this paradigm of self-reinforcing loops has
found consistent support from experimental findings in
the small-RNA-dependent epigenetic pathways of both
fungi and plants.
In S. pombe, the discovery that pericentromeric
H3K9 methylation is dependent on the RNAi machinery
was quickly followed by the realization that, conversely,
pericentromeric siRNA accumulation also requires the
H3K9 methyltransferase Clr4 (REFS 27,49). Several physi-
cal interactions are now known to underlie this mutual
dependence. First, the bivalent nature of the RITS com-
plex — its ability to interact with nascent RNA via its
siRNA-bound Ago1 subunit and with chromatin via
its Chp1 subunit — forms the physical basis for a self-
reinforcing positive feedback loop (FIG. 1). Methylated
H3K9 stabilizes RITS complex binding via Chp1 (REF. 49),
promoting siRNA amplification by the RITS complex-
interacting factors RDRC and Dcr1 (REFS 27,50). Second,
methylated H3K9 enhances siRNA accumulation by
helping to recruit the RDRC via the heterochromatin
protein 1 (HP1) homologue Swi6 in an interaction
that is directly bridged by Ers1 (REFS 30,51,52). Finally,
direct associations between the siRNA-programmed
RITS complex and the Rik1 and Stc1 subunits of the
CLRC38–40 ensure that siRNAs in turn feed back on H3K9
methylation states (FIG. 1).
The notion of self-reinforcing epigenetic states that
involve multiple types of signals was first proposed by
Eric Selker while working with the filamentous fungus
Neurospora crassa46. Initial results revealed that DNA
methylation patterns were dependent on histone
deacetylation, but recent findings suggest that feedback
loops that involve small RNAs also exist in N. crassa.
The canonical DNA methylation pathway is RNAi-
independent 53 and acts downstream of, and without
feeding back on, H3K9 methylation states54. By con-
trast, a newly uncovered DNA methylation phenom-
enon exhibits self-reinforcing properties and correlates
with the biogenesis of small RNAs. This type of DNA
methylation occurs at gene-rich and intergenic loci that
are transcribed in a convergent manner and that also
give rise to Dicer-independent siRNAs (disiRNAs)55,56.
Remarkably, disiRNA loci DNA methylation (DLDM),
unlike the well-studied DNA methylation pathway asso-
ciated with repetitive sequences, is required for H3K9
trimethylation (H3K9me3) and is thus self-reinforcing
through the known HP1‑dependent mechanism that
recruits the DNA methyltransferase DIM‑2 to the
regions of H3K9 methylation54,55. It was also observed
that, for a given DLDM locus, most clones are not meth-
ylated; however, when methylation does occur clones are
methylated extensively across the disiRNA-producing
locus, suggesting that a powerful positive feedback loop
leads to the spread of the epigenetic mark55. Convergent
transcription is critical and, when induced artificially, is
sufficient to drive this dynamic DNA methylation pat-
tern55. The potential roles of the disiRNAs themselves,
and other mechanistic aspects of DLDM, remain to
be elucidated, but these results further underscore the
prevalence of self-reinforcing epigenetic loops among
small-RNA silencing pathways in the nucleus.

RNA-directed DNA methylation in A. thaliana. In
A. thaliana, 24‑nucleotide siRNAs direct de novo DNA
methylation and maintenance of DNA methylation
at asymmetrical CHH sites (where H represents any
base except G) in a manner that depends on two plant-
specific polymerase II (Pol II)‑related RNA polymerases,
Pol IV and Pol V57 (reviewed in REF. 58). This phenom-
enon exhibits many physical connections between
pathway components that form the basis of a self-
reinforcing epigenetic loop (FIG. 2). First, 24‑nucleotide
siRNAs are generated by DCL3 processing of double-
stranded RNA (dsRNA) synthesized by the RdRP
RDR2 using Pol IV transcripts as templates. Recent
in vitro experiments have shown that RDR2 and Pol IV
associate in an RNA-independent manner and that the
activity of RDR2, but not of Pol IV, requires this associa-
tion, which suggests an evolved mechanism for limiting
siRNA biogenesis to loci occupied by Pol IV59. Loading
of siRNAs onto AGO4 in the cytoplasm triggers its
import into the nucleus60, where it associates directly
with siRNA-complementary Pol V transcripts61,62. This
sequence-specific recruitment is reinforced by direct
interactions between AGO4 and the GW domains of
both Pol V and the Pol V transcript-binding protein
KTF1 (also known as SPT5L)25,63. While these multiple
associations stabilize the localization of AGO4 along a
Pol V nascent transcript, the RDM1 protein provides
a physical link between AGO4 and the DRM2 DNA
methyltransferase, illustrating how the instruction
to silence gene expression is conveyed from small
RNAs to covalent DNA modifications64. RDM1 also
has an affinity for methylated DNA, which raises the
possibility that it can direct the Pol V–KTF1–DRM2–
AGO4 complex to pre-existing sites of DNA methyla-
tion. A recent study shows that AGO4 co‑purifies with
DRM2 in vivo, and that DRM2 preferentially methyl-
ates the strand of DNA that acts as a template for Pol V
transcripts65. The authors propose that as AGO4 binds
to a siRNA-complementary nascent Pol V transcript, it
directs DRM2 specifically to the template DNA strand as
it emerges from the Pol V exit channel. This study pro-
vides an unprecedented level of mechanistic detail on the
feedback between small RNAs and DNA methylation.
Interestingly, at a subset of loci, deletion of DNA
methylation by mutations in the histone deacetylase
HDA6 or the maintenance cytosine methyltransferase
MET1 results in loss of silent locus identity 66. This obser-
vation indicates that, at these loci, silent locus identity
is maintained by self-reinforcing interactions involv-
ing histone deacetylation and DNA methylation, while
siRNA amplification and silencing rely on cooperation
between the Pol V–KTF1–DRM2–AGO4 complex and
Pol IV machineries (see above) and on pre-existing
chromatin modifications66.
Embedded in the RNA-dependent DNA methylation
pathway are important self-reinforcing feedback mecha-
nisms that have been discovered only lately. For instance,
regions of RNA-directed DNA methylation in A. thaliana
are also enriched in H3K9 methylation58, and these two
epigenetic signals mutually support one another. Three
distinct H3K9 methyltransferases, KYP (also known
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as SUVH4), SUVH5 and SUVH6, contribute to the
maintenance of DNA methylation patterns, with KYP
having the primary role58. KYP is recruited to sites
of DNA methylation via its SET and RING finger-
associated (SRA) domain67 and, conversely, the Pol
IV‑associated factor SHH1 binds to nucleosomes
with methylated H3K9 and unmethylated H3K4
via its SAWADEE domain, thus promoting siRNA-
dependent methylation of the underlying DNA68,69.
Another example of self-reinforcement involves
the SUVH2 and SUVH9 proteins which, like KYP,
bind to methylated DNA via their SRA domains
but, unlike KYP, lack H3K9 methyltransferase activ-
ity 70. Two recent studies indicate that these SRA
domain-containing proteins directly recruit Pol V to
sites of pre-existing DNA methylation, thus generat-
ing nascent transcript scaffolds for recruitment of
AGO4–siRNA complexes and further enhancement
of the DNA methylation signal71,72. This dense network of
positive feedback in A. thaliana suggests that the broad
evolutionary relevance of self-reinforcing siRNA-driven
epigenetic loops extends beyond the S. pombe system in
which they were first suggested.
Conservation and divergence of nuclear RNAi
Below, we discuss examples of small-RNA-mediated
silencing in the animal germ line, including C. elegans,
D.  melanogaster and mammals. Nuclear RNAi in
these systems differs from the examples in S. pombe
or A. thaliana in one major aspect. Although animal
small RNAs act as signals that trigger the modifica-
tion of chromatin or DNA, it remains unclear whether
small-RNA amplification involves a self-reinforcing
Box 3 | Self-reinforcing positive feedback loops
Self-reinforcing positive feedback loops are formed
by the functional coupling of different types of signal
generation events. Studies in Schizosaccharomyces
pombe, Arabidopsis thaliana and mammals have
uncovered the molecular basis of these coupling
events by identifying the proteins that recognize
either histone H3 lysine 9 dimethylation (H3K9me2)
and H3K9me3, or DNA cytosine methylation
(DNA‑5mC), and that recruit enzymes which catalyse
the other methylation events. In some organisms (for
example, S. pombe and A. thaliana), the methylation
events are also physically coupled to proteins that
recruit small interfering RNA (siRNA) amplification
loops to chromatin, thus forming self-reinforcing loops
in which the histone or DNA methylation event
promotes siRNA generation and siRNAs in
turn promote histone or DNA methylation.
Self-reinforcing loops are thought to help to ensure
the maintenance of epigenetic information47,48.
H3K9 me2/3 siRNA H3K9 5mC
Nature Reviews | Genetics
REVIEWS

relationship with the downstream chromatin or DNA
modifications. Recent studies of the D. melanogaster
Piwi-interacting RNAs (piRNAs), so called because
they partner with Ago proteins of the largely germline-
specific Piwi subfamily, point in this direction, but
mammalian pathways seem to lack this characteristic.
Nuclear RNAi-related events associated with the intro-
duction of foreign siRNAs into animal somatic cells are
not discussed here.
Small RNAs and chromatin in animal cells: the piRNA
system. piRNAs were first discovered in mice and
D. melanogaster, and their roles in silencing transpo-
sons in the germ line of these organisms are well estab-
lished (reviewed in REF. 73). The C. elegans genome
also encodes piRNAs, called 21U‑RNAs, but they have
only been implicated in the repression of one transpos-
able element 74,75; we discuss the roles of 21U-RNAs in
epigenetic silencing in the next subsection.
In D. melanogaster, recent evidence suggests that
the nuclear protein Piwi, in addition to mediating
post-transcriptional gene silencing of transposons by
contributing to ‘ping-pong’ amplification with its cyto-
plasmic counterparts Aubergine (Aub) and Ago3, also
targets transposons at the transcriptional level76–80. In
ovarian somatic cells, which also harbour piRNAs, most
of the euchromatic H3K9 methylation islands in the
genome correspond to transposon insertion sites and
are Piwi-dependent 79. Furthermore, artificial recruit-
ment of Piwi to a reporter locus induces H3K9 meth-
ylation, HP1a (also known as Su(var)205) accumulation
and exclusion of RNA Pol II, suggesting that piRNAs
may have a direct role in guiding chromatin changes76,77.
Piwi and HP1a were previously reported to interact
directly in vivo 81, but the reproducibility and physio-
logical importance of this result have been debated77,80,
raising the possibility that there are unidentified links
that transmit instructions from piRNAs to chromatin.
Mechanisms involving Piwi engagement with nascent
transcripts or even the underlying DNA have been
proposed76,77, but understanding the molecular details
remains a major challenge for future research.

24-nt siRNA

DCL3

Acetylated nucleosome dsRNA Cytoplasmic loading followed by nuclear

import of AGO4–siRNA complex

RDR2
H3K9 methylated
nucleosome

GW
AGO4
DRM2
CMT3 RDM1 KTF1
GW

MET1
H3K9 methylated and
H3K4 unmethylated Pol IV Pol V
nucleosome
SHH1 SWD KYP SRA SUVH2
HDA6 and
Methylated DNA SUVH5 SUVH6 SUVH9

Figure 2 |  A self-reinforcing loop linking siRNAs to DNA and histone
methylation in A. thaliana.  Elaborate feedback between small RNAs and
DNA and histone methylation underlies a robust silencing pathway at sites
of asymmetrical cytosine methylation in the Arabidopsis thaliana genome.
Two plant-specific polymerases transcribe the critical RNAs. RNA
polymerase IV (Pol IV) transcripts are processed by the RNA-dependent
RNA polymerase RDR2 and the Dicer protein DCL3 into 24‑nucleotide (nt)
small interfering (siRNAs), while Pol V transcripts act as their targets. The
Argonaute protein AGO4, the siRNA-dependent recruitment of which to
Pol V transcripts is reinforced by interactions with the GW domains of Pol V
and an associated elongation factor KTF1, in turn recruits the CHH DNA
methyltransferase DRM2. RDM1 associates with the Pol V–AGO4–DRM2
complex and may link siRNA amplification to pre-existing DNA
methylation. Meanwhile, another DNA methyltransferase that targets CHG
sites for maintenance, CMT3, is recruited directly to methylated histone
H3 lysine 9 (H3K9). Silencing by DNA methylation is augmented by H3K9
methylation, which is deposited by the enzymes KYP, SUVH5 and SUVH6.
Nature
These methylation events are coupled to one another Reviews
and | Genetics
to siRNA activity
in several ways. KYP is recruited directly to methylated DNA, where it
methylates neighbouring histones, and the H3K9 methylation reader SHH1
recruits Pol IV to promote siRNA generation, while the DNA methylation
readers SUVH2 and SUVH9 recruit Pol V to promote AGO4 targeting and
further DNA methylation. Thus, the different methylation readers, RNA
polymerases and AGO4 act together to create self-reinforcing interactions
between pre-existing DNA methylation and siRNA amplification. Erasure
of DNA methylation by mutations in either the histone deacetylase HDA6
or the maintenance DNA methyltransferase MET1 results in loss of siRNA
biogenesis, emphasizing the importance of these self-enforcing
interactions. Altogether, the A. thaliana pathway for DNA methylation at
asymmetrical sites is one of the most remarkable examples of a recurring
theme in epigenetic regulation by small RNAs: self-reinforcing feedback
loops. SRA, SET and RING finger-associated; SWD, SAWADEE domain.

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© 2015 Macmillan Publishers Limited. All rights reserved


Long terminal repeat
(LTR). A DNA sequence that
is repeated at the ends of
retrotransposons or pro-viral
DNA that is formed from
retroviral RNA by reverse
transcription. Plant and
mammalian genomes contain
thousands of LTRs.
Epigenetic phenomena
Phenomena in which changes
in gene expression occur
without a corresponding
change in the DNA sequence;
such changes are stable in the
absence of initiating signals.
Paramutation
The ability of a silent allele to
convert an active allele to the
silent (and paramutagenic)
form. It was first described in
Zea mays.
NATURE REVIEWS | GENETICS VOLUME 16 | FEBRUARY 2015 | 77
Recent findings also show that sites bearing Piwi-
dependent H3K9 methylation in turn function as source
loci for piRNA production in D. melanogaster ovaries.
Methylated H3K9 recruits the HP1 family protein Rhino,
which, together with the Cutoff protein, promotes non-
canonical transcription from these loci to generate pre-
cursors to be processed into piRNAs82,83. Therefore, not
unlike the plant and fungal systems discussed above, the
D. melanogaster piRNA pathway seems to exhibit a posi-
tive feedback loop in which small RNAs guide histone
methylation, which in turn enables biogenesis of more
small RNAs.
In mice, piRNAs silence transposons in the male
germ line by targeting them for de novo DNA methyl­
ation during late embryonic and early neonatal devel-
opment 84–87. Consistently, the nuclear PIWI protein,
Piwi-like protein 4 (PIWL4; also known as MIWI2), is
expressed at this time85. Thus, like plants, mammals have
an RNA-directed DNA methylation pathway, although
few of its mechanistic details are currently understood.
Arguing against a self-reinforcing loop is the observation
that piRNA levels remain high in DNA (cytosine‑5-)-
methyltransferase 3‑like (Dnmt3l) mutants, in which
transposon DNA methylation is completely compro-
mised85. However, it is unclear to what extent H3K9
methylation at transposons, which could also contrib-
ute to piRNA generation, is affected in Dnmt3l mutants.
One study has suggested that piRNA-directed DNA
methylation in mice involves a system that is similar to
the nascent transcript model in S. pombe88. The authors
found that piRNAs mapped to a long terminal repeat
(LTR) adjacent to a paternally imprinted locus, RAS
protein-specific guanine nucleotide-releasing factor 1
(Rasgrf1), and that piRNA pathway components are
required for methylation of Rasgrf1. They also found
evidence of sequence-specific piRNA-dependent cleav-
age of a non-conding RNA overlapping with the LTR,
supporting the nascent transcript theory 88. However, it
has also been shown that the endonuclease activity of
PIWL4 is completely dispensable for DNA methylation
of long interspersed elements (LINE1) and the intracis-
ternal A particle (IAP) family of LTR retrotransposons89.
This suggests that nascent transcript slicing is not an
important event in small-RNA-dependent chromatin
silencing in mice, although it has a critical role in piRNA
amplification and post-transcriptional gene silencing.
Finally, piRNAs also contribute to epigenetic phenomena
in which they are themselves responsible for epigenetic
inheritance. This idea first emerged in a landmark
paper demonstrating the requirement for maternal
deposition of piRNAs, in addition to the presence of
genomic piRNA loci, for effective transposon silencing
and fertility in the offspring 15. A recent study found that
in D. melanogaster, strains containing certain P‑lacZ
transgene insertions, but not other strains containing
similar insertions at the same site, showed silencing
of homologous sequences in trans 90. Remarkably, if
maternally inherited, the trans-silencing-competent
alleles could convert the paternally inherited alleles
into strong trans-silencers, which was accompanied by
de novo production of piRNAs corresponding to these
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
trans-silencing loci. The new allele also acquired the
ability to convert other alleles into trans-silencers in
the same manner; thus, this constitutes a case of
paramutation90,91. These results indicate that maternally
deposited cytoplasmic piRNAs can direct the conversion
of homologous sequences into piRNA-generating loci
with trans-silencing ability. This work shows how a locus
can become incorporated into the piRNA repertoire and
how RNA itself can act as a carrier of epigenetic informa-
tion. Future discoveries regarding the molecular basis
of this paramutation process will provide fundamental
insights into both piRNA biology and the principles of
epigenetic inheritance.
RNAi and H3K9 methylation in C. elegans. C. elegans
has an array of 27 AGO homologues that associate with
diverse small RNA populations and that act in distinct
silencing steps92. Classical RNAi, induced by exogenous
dsRNA, initially involves the AGO protein RDE‑1 and
primary siRNAs; however, RdRPs generate secondary
siRNAs that are not loaded onto RDE‑1 (REF. 92). Among
other roles, secondary siRNAs can trigger RNAi in the
nuclei of somatic cells by acting with the AGO protein
NRDE‑3. Upon small-RNA loading, NRDE‑3 enters
the nucleus, associates with complementary nascent
transcripts and recruits the non-AGO silencing factor
NRDE‑2, which in turn promotes H3K9 methylation
and inhibits elongation of RNA polymerases beyond
the site targeted by siRNAs17,93,94 (FIG. 3). Thus, as in
fungi and plants, small RNAs in C. elegans can trigger
transcriptional gene silencing. However, currently, the
biochemical connections remain obscure except for
a direct interaction between NRDE‑3 and NRDE‑2
(REF. 16). More recent work has shown that additional
AGO homologues are also required for chromatin
targeting 17. Interestingly, RNAi-induced H3K9 methyl­
ation is a transgenerational phenomenon, as it is inherited
for at least two generations, in the absence of the
dsRNA trigger, by a mechanism that involves germline
transmission of siRNAs17,95.
In the C. elegans germ line, inheritance of H3K9
methylation induced by exogenous dsRNA is mediated
by the germline-specific AGO protein HRDE‑1 (also
known as WAGO‑9) in association with RdRP-derived
siRNAs96,97. Intriguingly, both HRDE‑1 and NRDE‑2
are also necessary for germline immortality and fertil-
ity 97. This biological requirement is reminiscent of the
piRNA pathway, the widely conserved germline main-
tenance system discussed above and represented in
C. elegans by the 21U‑RNAs and the PIWI-subfamily
AGO protein PRG‑1 (REF. 74). Indeed, several studies
have concluded concurrently that HRDE‑1 is in fact the
downstream effector of piRNA-induced silencing in
the germ line96,98–100. Another observation was also made
— while HRDE‑1, NRDE‑2 and the HP1 homologue
HPL‑2 are required to maintain a permanent memory
of these silencing events at the chromatin level, PRG‑1
and 21U‑RNAs are only required for its initiation96,99,100
(FIG. 3). Thus, different components are responsible for
the establishment and maintenance steps in this example
of RNA-mediated epigenetic regulation.
REVIEWS

a Somatic nuclear RNAi pathway b Germline non-self and self pathways

Non-self RNA Self RNA
Dicer and PRG-1
loading factors U

RDE-1
RdRP
G
G
G G
Post-transcriptional 22G-RNA
gene silencing G

RdRP RDE-1
HRDE-1 CSR-1
AAA G G

NRDE-3
G
Cytoplasm
Nucleus

NRDE-3 HRDE-1 –

NRDE-2 – NRDE-2 CSR-1

HMTase HPL-2 HMTase HPL-2

Pol II Pol II
H3K9me3

Figure 3 |  Small-RNA-driven transcriptional silencing of gene expression in C. elegans.  a | In Caenorhabditis

Nature Reviews | Genetics
elegans, exogenous double-stranded RNA (dsRNA) is processed into primary small interfering RNAs (siRNAs) that
are loaded onto the Argonaute (AGO) protein RDE‑1 and amplified by RNA-dependent RNA polymerases (RdRPs)
to give rise to secondary siRNAs called 22G‑RNAs. When loaded with 22G‑RNAs, the somatic AGO protein NRDE‑3
translocates to the nucleus, where it targets nascent RNA transcripts and silences corresponding genes, acting in
concert with the silencing factor NRDE‑2. Gene expression is halted by NRDE‑2 during the elongation phase of
transcription, and silencing involves histone H3 lysine 9 trimethylation (H3K9me3) and recruitment of the
heterochromatin protein 1 (HP1)‑like protein HPL‑2. b | In the germ line, small-RNA-directed transcriptional
silencing is mediated not by NRDE‑3 but by a different AGO protein, HRDE‑1, which also acts through NRDE‑2,
H3K9 methylation and HPL‑2. HRDE‑1 receives 22G‑RNA inputs both from the pathway that responds to
exogenous dsRNA and from the PIWI-interacting RNA (piRNA), or 21U‑RNA, pathway that scans the transcriptome
for foreign RNAs. 21U‑RNA-programmed PRG‑1 promotes the RdRP-dependent generation of 22G‑RNAs, which
are loaded onto HRDE‑1. In both cases, HRDE‑1 maintains a persistent, transgenerational memory of silenced
genes in the germ line. Meanwhile, another AGO protein called CSR‑1 binds to 22G‑RNAs that represent the full
complement of endogenously expressed RNAs, and protects the corresponding loci from possible silencing by
HRDE‑1. Thus, the 22G‑RNAs bound by CSR‑1 and HRDE‑1 transmit a germline memory of ‘self’ and ‘non-self’
RNAs, respectively, to be appropriately licensed for expression or silenced. HMTase, histone methyltransferase;
Pol II, RNA polymerase II.

In contrast to piRNAs in D. melanogaster and mam-
mals, C. elegans piRNAs only silence one transposon
family 74,75. However, transgene insertions can be silenced
in a PRG‑1‑dependent manner 96,99,100, supporting a con-
served function for piRNAs in recognizing ‘non-self ’
nucleic acids. Interestingly, PRG‑1 was found to initiate
epigenetic silencing of the same single-copy transgene
in some lines but not others100. Genetic crosses indicate
that transgene expression states can be converted in
both directions by trans-acting silencing and activating
factors, leading to the proposal that HRDE‑1–siRNA
complexes enforce the maintenance of PRG‑1‑directed
silencing, whereas another system prevents or even
reverses its establishment 100. The suggested candidate
is the AGO protein CSR‑1, the RdRP-dependent small
RNA partners of which map not to silenced loci but
instead to all mRNAs expressed in the germ line101.
This was confirmed by recent experiments in which
CSR‑1‑dependent activity was shown to counteract
PRG‑1‑mediated silencing in a heritable manner 102,103.
Thus, PRG‑1, associated with 21U‑RNAs, is proposed
to sample cellular transcripts for non-self sequences to
be silenced by HRDE‑1, while CSR‑1 carries a reper-
toire of self-expressed sequences to be protected from
silencing 96,99,100,102,103 (FIG. 3). CSR‑1 seems to have a criti-
cal role in sperm, where it promotes spermiogenic gene
expression and where its associated small RNAs reflect
previous germline expression patterns104.

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© 2015 Macmillan Publishers Limited. All rights reserved


RNA-dependent histone methylation without RNAi
In the RNAi-mediated chromatin silencing pathways
discussed above, a recurring feature is the role of RNA
as an assembly scaffold. Below, we focus on analogous
but RNAi-independent strategies used by both mRNAs
and lncRNAs to recruit histone-modifying enzymes.
LncRNAs, typically defined as transcripts more than
200 nucleotides in length that do not encode proteins,
have been uncovered in large numbers as a result of
rapid advances in high-throughput technologies. The
function of lncRNAs in genome regulation, although still
largely unknown, seems to be diverse and is the sub-
ject of a growing field of study (reviewed in REF. 105). A
common theme in current models is the ability of many
lncRNAs to target chromatin-modifying activities to
particular genomic sites. Perhaps the most extensively
studied examples are those that involve dosage compen-
sation in metazoans (reviewed in REF. 106), including
the mammalian lncRNA XIST, which mediates global
inactivation of a randomly chosen X chromosome in
females in a process known as X chromosome inacti-
vation (XCI). XIST has been proposed to function by
directing the Polycomb repressive complex 2 (PRC2)
H3K27 methyltransferase to chromatin. Below, we
examine the evidence and unresolved questions regard-
ing the recruitment of Polycomb to chromatin by XIST
and other lncRNAs. The basis of specificity for the pro-
posed protein–RNA interactions remains unknown.
We also discuss a class of lncRNAs transcribed from
enhancers that are thought to activate gene expression
in cis. Finally, we describe a case in which the mode of
specificity is better understood — the targeting of the
Clr4 H3K9 methyltransferase by the mRNAs of meiotic
genes to their gene loci in S. pombe.
lncRNAs and Polycomb recruitment. Polycomb pro-
teins and H3K27me3 were first linked to XCI by genetic
and cytological experiments107–109. A more direct bio-
chemical connection was shown by the detection of
XIST RNA in PRC2 immunoprecipitation experi-
ments and a corresponding in vitro test of their inter-
action110. REPA — an independent internal transcript
within XIST, so called because it spans the conserved
A repeat region of XIST — was proposed to interact
directly with and recruit the PRC2 catalytic subunit
EZH2 in the earliest stages of XCI establishment. The
same region of full-length XIST would then directly
recruit PRC2 as silencing spreads in cis110. It was also
reported that other lncRNAs—such as KCNQ1 opposite
strand/antisense transcript 1 (Kcnq1ot1) and HOX
transcript antisense RNA (HOTAIR)—could directly
recruit PRC2 to chromatin in cis or in trans, respec-
tively111,112. However, one study failed to detect an inter-
action between PRC2 and the XIST A repeats using an
ultraviolet crosslinking approach113, and others have
found the XIST A repeat to be dispensable for PRC2
chromatin association and H3K27 methylation in mice
and humans107,114. More generally, the idea that lncRNAs
mediate PRC2 targeting through direct interactions has
been undermined both by doubts regarding specificity
and by the existence of equally compelling alternative
NATURE REVIEWS | GENETICS VOLUME 16 | FEBRUARY 2015 | 79
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
scenarios (reviewed in REF. 115). The number of RNAs
found to be associated with PRC2 in cells is enor-
mous116–119, highlighting the risk of interpreting indi-
vidual interactions as specific. For instance, a recent
study revealed that the binding affinity of PRC2 for the
bacterial MBP mRNA is equal to its binding affinity for
HOTAIR, and that RNA length is a far better predictor of
PRC2 binding affinity than RNA sequence116. Thus, the
direct interactions reported between PRC2 and lncRNAs,
such as HOTAIR, RepA and Kcnq1ot1 (REFS 110–112)
among others, offer only minimal insight into the regu-
lation of methyltransferase recruitment to particular
genomic loci.
Although the currently prevailing models concerning
Polycomb recruitment by lncRNAs remain inconclusive,
the reality is likely to involve a much subtler and more
complex system of sequentially and/or cooperatively
assembled components, in which specific lncRNAs act
as scaffolds. For example, the Jumonji family protein
JARID2 was recently reported to act as an essential inter-
mediate between Xist and PRC2 (REF. 114). While the A
repeat of Xist is required for gene silencing, it does not
seem to be required for the recruitment of PRC2, as pre-
viously proposed. Instead, Xist recruits JARID2 via its B
and F repeats, and JARID2 is required for the localization
of PRC2 and methylation of H3K27 (REF. 114). Another
study has suggested that the facilitation of JARID2–
PRC2 interactions may represent a more general func-
tion of lncRNAs120, although the amino acid residues that
were thought to mediate JARID2 RNA binding differ
between the two studies114,120. Thus, the events linking
lncRNAs to the recruitment of PRC2, and other meth-
yltransferases to chromatin, are likely to be multiple
and intricate, involving the contributions of other pro-
tein factors and perhaps other histone modifications115.
The increasing scepticism towards widespread assump-
tions is promising and should ultimately lead to a better
understanding of how lncRNAs influence chromatin.
lncRNAs and enhancer function. Transcription at a large
number of mammalian enhancers gives rise to lncRNAs,
called enhancer RNAs (eRNAs), that seem to have major
roles in the regulation of transcription. At least a subset
of eRNAs seem to act as nascent transcripts that func-
tion in cis to promote the transcription of neighbour-
ing genes. Initially, high-throughput RNA sequencing
experiments identified thousands of eRNAs that are
transcribed from enhancer elements in response to sig-
nals that mediate enhancer-dependent transcriptional
activation121,122. RNAi-mediated knockdown of several
eRNAs was then shown to result in reduced expression
of proximally located target mRNAs, suggesting that
eRNAs act in cis and are required for the activation of
target gene transcription123,124.
Insights into the mechanism of action of eRNAs
came from the identification of components of the
transcription machinery that mediate eRNA function.
eRNAs involved in the activation of the developmen-
tally regulated genes T-cell acute lymphocytic leukae-
mia 1 (TAL1), snail family zinc-finger 1 (SNAI1) and
SNAI22 require the Mediator transcription co‑activator
REVIEWS

a activation. Large RNAs (>200 nucleotides) may be par-

DNA-binding Histone or DNA
proteins E
methylation
b cussed here but have recently been extensively reviewed
AGO or PIWI
E elsewhere22,127.
Histone or DNA
methylation
Pol II
c removal (DSR)128. Remarkably, DSRs are found within the
RNA-binding
protein
E they must exhibit some degree of sequence flexibility to
Histone or DNA
methylation ? of different variants of a hexanucleotide motif 129. At a
Pol II
Figure 4 |  RNAs, both short and long, represent an alternative to DNA-binding
Nature Reviews | Genetics
proteins as specificity determinants for epigenetic regulation of gene expression.  How do DSR-containing mRNAs recruit the Clr4
Enzymes (E) that catalyse methylation of histone tails or cytosine bases in DNA are
recruited to chromatin by distinct mechanisms. a | Sequence-specific DNA-binding
proteins recruit histone- or DNA-modifying enzymes to chromatin. b | Small RNAs
target an Argonaute (AGO) or PIWI protein to a nascent transcript through
base-pairing interactions to recruit modifying enzymes. c | Long RNAs act as scaffolds
for RNA-binding proteins to recruit chromatin-modifying complexes. In all cases, the
binding of the enzyme or the recruiting factors (for example, AGO–PIWI complexes in
part b and RNA-binding protein complexes in part c) to chromatin may be enhanced
by interactions with pre-existing modifications, which self-reinforce the epigenetic
state. Pol II, RNA polymerase II.
for their activity 125, while HOXA distal transcript anti-
sense RNA (HOTTIP), an eRNA involved in the activa-
tion of HOXA homeobox genes, functions through the
WDR5–MLL (also known as KMT2A) H3K4 methyl-
transferase complex 124. Chromosome conformation cap-
ture experiments show that the eRNA mediates looping
interactions between the enhancer and promoter regions
of genes. These studies have given rise to an attractive
model in which eRNAs recruit co‑activator complexes
and promote their interaction with gene promoters to
activate transcription. Consistent with this model, there
is evidence that supports a role for specific enhancer
sequences that encode eRNAs, which are different from
enhancer sequences that form binding sites for transcrip-
tion factors, in activation of target genes126. These studies
provide a new function for nascent RNA transcripts as
scaffolds for the recruitment of co‑activator complexes
that mediate chromosome looping and transcriptional
ticularly suited for such architectural tasks that bring
enhancer and promoter regions, usually located great
distances apart, into proximity.
The identification of both the regions within eRNAs
that mediate co‑activator complex recruitment and the
RNA-binding domains in the subunits of these com-
plexes is required for a better understanding of how
different eRNAs activate transcription. In addition to cis-
acting eRNA, several trans-acting lncRNAs that activate
transcription have also been identified. They are not dis-
Silencing of meiotic genes in S. pombe. The regulation of
certain meiotic genes in S. pombe is another well-studied
example in which an RNA scaffold promotes the RNAi-
independent recruitment of a histone methyltransferase
to chromatin. Meiosis-specific genes are silenced in veg-
etative cells by a post-transcriptional process that tar-
gets an RNA cis-element called determinant of selective
protein-coding regions of meiotic mRNAs128. Therefore,
preserve meiotic protein structure but effectively consist
subset of DSR-containing genes, notably mei4+ and ssm4+,
H3K9 methylation is also observed and depends on the
DSR and transcription, which suggests that the mRNAs
of these genes direct Clr4 to chromatin and may promote
transcriptional and post-transcriptional silencing 130,131.
methyltransferase? Much like lncRNAs and Polycomb
proteins, the process seems to involve a host of factors
rather than direct interactions between the RNA and
the histone-modifying enzyme. In the case of meiotic
mRNAs, the source of specific recognition is much
clearer. DSRs are bound directly by the YTH-domain
protein Mmi1, and this binding event is required for mei-
otic gene silencing and for H3K9 methylation of the mei4+
and ssm4+ loci128,129,131. The Mmi1‑interacting zinc-finger
protein Red1 is also required for silencing and H3K9
methylation at mei4+ and ssm4+ (REFS131,132). Strikingly,
Red1 associates with Clr4, which suggests that it serves as
the critical link that targets histone methylation to genes
producing DSR-bearing transcripts130,131. Interestingly, this
Mmi1–Red1–Clr4 axis of RNA-directed H3K9 methyl­
ation also seems to operate outside the meiotic process
to target context-specific genes such as pho1+, which is
repressed when phosphate is available. Data from two
recent studies show that an upstream lncRNA mediates
Red1 chromatin binding and H3K9 methylation at the
pho1+ locus in a phosphate- and Mmi1‑dependent man-
ner 133,134. Importantly, the lncRNA was found to contain
DSR motifs that are critical for this regulation134.
While the DSR-bearing mRNAs demonstrate RNA-
mediated recruitment of histone modifications with-
out RNAi, silencing itself seems to be independent of
H3K9 methylation. This is perhaps made most evident
by the observation that some DSR-containing genes are
silenced by Mmi1 and Red1 without exhibiting H3K9

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 REVIEWS

methylation 131. DSRs inserted ectopically in other
genes produce similar effects 130. Most conclusively,
cells lacking Clr4 show wild-type silencing of all tested
meiotic genes during vegetative growth 135. Instead,
the critical event for silencing of these genes seems to
involve degradation by the nuclear exosome through
a mechanism that requires polyadenylation of the tar-
get RNAs136. Consistently, Red1 interacts with the exo-
some subunit Rrp6 (REF. 132), and proteomic analysis
recently showed that this association is mediated by the
Mtr4‑like helicase Mtl1, another recently identified Red1
interactor 133,135. This poses the question of what fitness
advantage is conferred by H3K9 methylation at meiotic
genes, if it is not necessary for silencing. The presence
of H3K9 methylation might reflect an ancestral path-
way that predates the contribution of the exosome and
that has not yet been lost 135. It is also possible that H3K9
methylation could control expression of DSR-containing
genes under certain circumstances, for example, during
gene induction134,135. Additional work is needed in this
area not only to define the biological function of histone
methylation but also to further isolate the steps that lead
to Clr4 recruitment, for example, to determine whether
Mmi1 and Red1 accomplish this task independently.
Studies of the meiotic mRNAs in S. pombe have pro-
vided a valuable paradigm for investigating the RNA-
mediated targeting of histone methyltransferases in all
its mechanistic complexity. In this regard, it is worth
noting that the XIST RNA is thought to have evolved
from a protein-coding gene137. This XIST precursor
gene may have shared certain features of the S. pombe
meiotic RNAs that enabled it to locally recruit repressive
chromatin-modifying complexes before it acquired the
ability to spread along the entire X chromosome.
Conclusions and perspectives
The mechanisms by which coding RNAs and non-
coding RNAs regulate chromatin structure in different
organisms share key similarities that allow us to note
common principles which unify these ancient path-
ways (FIG. 4). The first is the principle of recruitment via
nascent RNA. In these systems, recruitment of effector
complexes that methylate histones or DNA involves the
association of small-RNA-guided AGO complexes or
site-specific RNA-binding proteins with nascent coding
RNA or non-coding RNA scaffolds (FIG. 4b,c), rather
than specific sites on DNA and DNA-binding proteins
(FIG. 4a). The second major principle involves the role of
RNA as a component of self-reinforcing positive feed-
back loops. These loops are unique to small-RNA sys-
tems that contain an amplification component and have
key roles in the epigenetic inheritance of histone and
DNA methylation patterns. The key event is the locali-
zation of the small-RNA amplification machineries
on nascent transcripts, and their activation by the his-
tone or DNA methylation events induced by the small
RNAs themselves (BOX 2). Thus, in both S. pombe and
A. thaliana, small-RNA amplification and histone or
DNA methylation are co‑dependent. Self-reinforcing
loops have not yet been described for lncRNAs that act
independently of RNAi. However, cooperative recruit-
ment involving the association of chromatin-modifying
complexes with both pre-existing chromatin modifica-
tions and DNA-binding specificity factors has been sug-
gested to contribute to the maintenance of epigenetic
states in budding yeast 32. By analogy, it is possible that
direct or indirect interactions between RNA-binding
proteins that recognize lncRNA and also pre-existing
histone modifications help to reinforce lncRNA-medi-
ated changes in chromatin structure. Finally, small
RNAs, and their association with positive feedback loops
in the germ line, allow them to act as components of
transgenerational inheritance mechanisms. The trans-
mission of small RNAs through meiosis seems to act as a
signal for the inheritance of internal or environmentally
induced changes from parent to offspring 138,139.
Despite the considerable progress outlined in this
Review, several important questions about the biogenesis
and function of non-coding RNAs remain unanswered.
The mechanisms that distinguish between different
types of transcription and that trigger the generation of
different classes of small RNAs remain to be fully under-
stood, although the available evidence indicates a major
role for RNA processing events that act co‑transcription-
ally to determine whether a nascent transcript becomes a
functional mRNA or is marked for processing by RNAi
and other surveillance mechanisms. Finally, the mecha-
nisms by which lncRNAs participate in the recruitment
of Polycomb proteins and other chromatin-modifying
activities, particularly the molecular basis of specificity,
remain poorly defined. We can look forward to answers
to these questions and, if the recent past is a guide, to
more exciting and unexpected discoveries about the
roles of RNA in gene regulation.

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Acknowledgements
D.H. was supported by the US National Science Foundation
Graduate Research Fellowship Program. Research in D.M.’s
laboratory is supported by grants from the US National
Institutes of Health. D.M. is an Investigator of the Howard
Hughes Medical Institute.
Competing interests statement
The authors declare no competing interests.
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